Mechanistic understanding and inhibition of Zika NS5 and SARS-CoV-2 RdRP proteins ABSTRACT Zika virus (ZIKV) and Coronavirus (CoV) are single-stranded RNA viruses that pose grave threat to public health. In the first two decades of the 21st century, the global community has already witnessed one outbreak of Flavivirus, Zika virus (ZIKV), and three zoonotic outbreaks of CoV? severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in 2002, the Middle East respiratory syndrome (MERS)-CoV in 2012, and the most recent, the novel SARS-CoV-2. These viruses are highly transmissible, greatly impacting the social, societal and economic dynamics. However, there are currently no approved drugs for either ZIKV or for zoonotic CoV, raising an urgent need for development of novel therapeutic strategies against ZIKV and CoV infection. This application seeks to develop an antiviral strategy targeting the viral core replication machinery, RNA-dependent RNA polymerase (RdRP), non-structural protein 5 (NS5) of ZIKV and non- structural protein 12 (NSP12) of SARS-CoV-2. On one hand, the currently identified small molecule inhibitors will be evaluated for their efficiency on ZIKV or SARS-CoV-2 inhibition. On the other hand, mechanistic details of ZIKV NS5 and SARS-CoV-2 RdRP-mediated RNA replication will be investigated, thereby providing a basis for development of synergistic inhibition strategies targeting various enzymatic steps of ZIKV NS5 and SARS- CoV-2. In Aim 1, structural, biochemical and cellular approaches will be taken to evaluate the inhibition of ZIKV NS5- or SARS-CoV-2 RdRP-mediated de novo RNA synthesis by candidate inhibitors. Through evaluation of the inhibitory effects of the candidate inhibitors on ZIKV NS5 or SARS-CoV-2 RdRP, this application will address whether these compounds can serve as inhibitors to ZIKV NS5 or SARS-CoV-2, and more importantly, to provide a basis for structure-based drug optimization for ZIKV NS5 or SARS-CoV-2. In Aim 2, the mechanistic basis of ZIKV NS5 and SARS-CoV-2 RdRP-mediated RNA replication will be determined through structure elucidation of the replication complexes of ZIKV NS5 or SARS-CoV-2 RdRP, combined with enzymatic analyses. The structural knowledge on the replication complexes of ZIKV NS5 and SARS-CoV-2 RdRP will then provide a framework for structure-based drug design for comprehensive inhibition of ZIKV NS5 and SARS-CoV-2 infection. Together, the proposed studies will provide key mechanistic insights into the viral RdRP-mediated genome replication and establish a foundation for development of effective inhibitors against ZIKV and SARS-CoV-2.